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Abstract

Background

Diagnosis of ovarian carcinoma is in urgent need for new complementary biomarkers
for early stage detection. Proteins that are aberrantly excreted in the urine of cancer
patients are excellent biomarker candidates for development of new noninvasive protocol
for early diagnosis and screening purposes. In the present study, urine samples from
patients with ovarian carcinoma were analysed by two-dimensional gel electrophoresis
and the profiles generated were compared to those similarly obtained from age-matched
cancer negative women.

Results

Significant reduced levels of CD59, kininogen-1 and a 39 kDa fragment of inter-alpha-trypsin
inhibitor heavy chain H4 (ITIH4), and enhanced excretion of a 19 kDa fragment of albumin,
were detected in the urine of patients with ovarian carcinoma compared to the control
subjects. The different altered levels of the proteins were confirmed by Western blotting
using antisera and a lectin that bind to the respective proteins.

Conclusion

CD59, kininogen-1 and fragments of ITIH4 and albumin may be used as complementary
biomarkers in the development of new noninvasive protocols for diagnosis and screening
of ovarian carcinoma.

Background

Ovarian carcinoma is the leading cause of death among gynaecologic malignancy. It
is the fourth most common cancer affecting women in Malaysia [1]. Patients with ovarian carcinoma often presented themselves at an advance stage of
cancer mainly because of the lack of biomarker for early diagnosis and that the cancer
is usually asymptomatic at the early stages [2]. Once the cancer is detected at the advance stage, the five-year survival rate of
the patients decreases to 25% even when appropriate treatments were provided [3,4].

The gel-based proteomic analysis provides a convenient method to compare the levels
of proteins in bodily fluid samples. In the search for new protein biomarker candidates
with clinical diagnostic value, substantial progress has been made in the proteomic
analysis of serum samples of patients with different cancers [5-7]. In contrast, fewer studies have been carried out on the urine samples of cancer
patients. This is despite that urine is generally a better sample for investigative
and screening purposes and that the use of urine protein biomarkers such as albumin
and human chorionic gonadotropin for clinical diagnosis has been a long standing practice.

The proteomic analysis of urine offers ample opportunities for clinical translation
[8,9]. To date, proteomic experiments that have been conducted on urine were not confined
to patients suffering from diseases of the genitourinary system [10] but were also carried out on those with atherosclerosis [11], sleep disorder [12] and cancers of the bladder [13], pancreas [14,15], lung [16] and colon [17]. Proteomic investigation has been performed on urine of patients with ovarian carcinoma
but is currently restricted to the low molecular weight peptide analysis using the
SELDI-TOF-MS approach [18].

In the present study, urine protein samples from patients with ovarian carcinoma and
cancer negative women were subjected to the conventional two-dimensional electrophoresis
(2-DE) and densitometry analysis. Proteins that were aberrantly excreted by the cancer
patients, relative to control subjects, were identified by mass spectrometry and their
altered levels in the patients urine were confirmed by Western blotting using antisera
and a lectin that bind to the respective proteins.

Results

2-DE profiles of urine proteins

Separation of urine protein samples by 2-DE resulted in highly resolved profiles comprising
more than ten clusters of protein spots. Panel A of Figure 1 demonstrates a representative urinary proteome profile obtained from a control subject.
Seven protein spot clusters consistently appeared in all the 15 control samples analyzed
and there was no apparent difference in the intensity of the spots between the individual
urine samples studied. When the gel-based proteomic analysis was performed on urine
protein samples from patients with ovarian carcinoma (n = 11), different 2-DE profiles
were obtained (Figure 1, panel B). Three protein spot clusters which consistently appeared in the control
profile were either not detected or were reduced in intensity in the cancer patients
while one protein spot appeared enhanced in a considerable number of the patients'
2-DE gels. The levels of the other protein spot clusters were comparable to those
detected in the urinary proteome profiles of the control subjects.

Figure 1.Typical 2-DE urine protein profiles of controls and patients with ovarian cancer. Panels A and B demonstrate the representative 2-DE urine protein profiles of control
subjects and ovarian cancer patients, respectively. The aberrantly excreted urine
protein spot clusters were marked in circles. KNG1, ITIH4f and ALBUf refer to kininogen-1
and fragments of inter-alpha-trypsin inhibitor heavy chain H4 and albumin, respectively.
Acid side of 2-DE gel is to the left and relative molecular mass declines from the
top.

Identification of aberrantly excreted urine proteins

Subjecting the spot clusters of urine proteins that were aberrantly excreted to mass
spectrometry and database search identified them as CD59, kininogen-1, inter-alpha-trypsin
inhibitor heavy chain H4 (ITIH4) and albumin. Table 1 shows a summary of the data acquired. High probability-based MOWSE scores were obtained
for all the urine proteins. Among the four urine proteins of interest, ITIH4 and albumin
demonstrated large discrepancies between the experimental masses that were estimated
based on their mobilities in the 2-DE gels and their theoretically calculated mass.
This suggested that the ITIH4 and albumin spots detected in the 2-DE urinary profiles
were truncated fragments of their native molecules.

In the case of ITIH4 (Q14624), the peptide sequences identified with high confidence
from the MS/MS correlated to the C-terminal region of the protein, when they were
checked against the Swiss-Prot database (Table 2). Sequences obtained were those that spanned within the kallikrein-generated 35 kDa
fragment region of ITIH4 (amino acids 696-930). However, molecular mass estimation
based on its relative mobility in 2-DE gels indicated a larger fragment of approximately
39 kDa. In case of albumin (P02768), the sequences derived from the MS/MS analysis
were confined to amino acids 118 to 281 of the molecule (Table 2).

Image analysis of 2-DE gels

The different altered levels of CD59, kininogen-1 and fragments of ITIH4 and albumin
in the urine of patients with ovarian carcinoma, relative to the controls, was confirmed
when their 2-DE urine protein profiles were subjected to image analysis using the
Image Master 2 D Platinum Software 7.0. Image analysis also confirmed that the levels
of the other highly resolved urine protein spot clusters were comparable between cancer
patients and controls. Figure 2 demonstrates the mean percentage of volume contribution of the four urine proteins
of interest in control subjects and patients with ovarian carcinoma. When taken as
overall, the levels of CD59, kininogen-1 and ITIH4 fragment were significantly lower
in ovarian carcinoma patients by 3.6-, 2.5- and 1.9-folds, respectively, compared
to those excreted by the control subjects. In contrast, the 19 kDa fragment of albumin
appeared 274-fold higher in the patients urine (Table 3).

Figure 2.Relative excretion of urine proteins by control subjects and patients with ovarian
cancer. The percentage of volume contribution was determined using the Image Master 2D Platinum
Software 7.0. Image analysis performed on protein spot clusters that appeared consistently
within each cohort of urine samples demonstrated the aberrant excretion of CD59, kininogen-1
(KNG1), ITIH4 (39 kDa fragment) and 19 kDa fragment of albumin (ALBUf) by patients
with ovarian carcinoma (OCa).

SDS-polyacrylamide gel electrophoresis and Western blotting

Further confirmation of the altered levels of CD59, kininogen-1 and fragments of ITIH4
and albumin in the urine of patients with ovarian carcinoma relative to those of the
control subjects was performed using antibodies and a lectin that bind to the respective
proteins that were blotted onto membranes. Figure 3 demonstrates the respective interactions of specific antibodies and the CGB lectin
with the four proteins of interest in pooled urine samples of patients and control
subjects. In case of the 19 kDa albumin fragment, interaction appeared to be detected
only in the pooled urine of patients with ovarian carcinoma compared to that of the
controls, while the inverse was observed for CD59, kininogen-1 and the 39 kDa fragment
of ITIH4.

Figure 3.Interaction of antisera and CGB lectin with aberrantly excreted urine proteins. Pooled urine samples of ovarian cancer patients (OCa) and those of control subjects
(Con) were subjected to SDS-PAGE and Western blotting before being independently exposed
to antisera that bind to CD59, kininogen-1 and albumin as well as the ITIH4 binding
CGB lectin.

Discussion

In the present proteomic profiling study, the significant reduced excretion of CD59,
kininogen-1 and a 39 kDa fragment of ITIH4, and the enhanced levels of a 19 kDa fragment
of albumin were detected in the urine samples of patients with ovarian carcinoma relative
to those of the control subjects. Their different altered levels in the urine of ovarian
cancer patients were confirmed by Western blotting using antisera and a lectin that
bind to the respective proteins. These urinary proteins have potential to be used
as complementary molecular indicators for noninvasive diagnoses and/or monitoring
of ovarian carcinoma, although this requires further confirmation involving a larger
scale clinical investigation.

CD59, a cell surface molecule, functions to inhibit the membrane attack complex of
the complement pathway. The soluble form of CD59 is usually found in normal human
urine at a concentration of about 3.7 μg/ml. However, it is barely detectable in the
blood (between 33-119 ng/ml), and even that only in the presence of detergents [19,20]. To the best of our knowledge, the present study is the first to report the decreased
levels of CD59 in the urine of patients with ovarian cancer although similar reduced
excretion of the protein had previously been reported in the urine of patients with
bladder cancer [13] and pancreatic ductal adenocarcinoma [14]. The reason for the low levels of CD59 in the urine of cancer patients is not understood.
One possibility is that since the turnover of cancer cells bearing CD59 is low as
they are generally "immortal", less of the cell surface molecules are being solubilized
and excreted in the urine. However, this remains to be further proven.

Like CD59, kininogen-1 is also detectable in the urine of healthy individuals. Previous
studies performed on serum and plasma samples have shown that the expression of kininogen-1
was significantly reduced in patients with gastrointestinal cancer [21], breast cancer [22] and two different types of cervical cancer [23]. Since kininogen-1 is known for its antiangiogenic properties and inhibitory action
on the proliferation of endothelial cells [24], its lowered expression in serum/plasma of the cancer patients was believed to have
contributed to the survival of the cancer cells [23]. In view of these previous reports, it was not surprising to find similar reduced
levels of kininogen-1 in the urine of patients with ovarian carcinoma in this study.
However, the aberrant kininogen-1 expression is apparently not cancer-specific since
decreased levels of the protein had previously been reported in the urine of patients
with chronic pancreatitis [14], interstitial cystitis [25] and IgA nephropathy [26], although the cause for the altered levels of kininogen-1 in these diseases may have
been different.

The precise reason for the reduced levels of the ITIH4 fragment in the urine of patients
with ovarian carcinoma that is observed in this study is currently not understood.
The estimated molecular mass of the urine ITIH4 fragment indicated that it was slightly
larger than its reported 35 kDa serum counterpart in the ovarian carcinoma patients
[27]. Detection of the different sizes of ITIH4 fragments was not surprising as previous
studies using SELDI-TOF-MS have demonstrated that ITIH4 was extensively processed
within its proline-rich region in the human serum. In different diseases including
ovarian carcinoma, different fragments were shown to be proteolytically generated
[28]. While the present study demonstrated the reduced levels of the 39 kDa ITIH4 fragment
in the urine of patients with ovarian carcinoma, our previous data showed the up-regulated
levels of a 35 kDa ITIH4 fragment in the serum samples of the patients [27]. This inverse relationship and the difference in the molecular masses of the ITIH4
fragments detected in the respective samples suggest presence of a selective glomerular
filtration mechanism that retained the 35 kDa fragment in the blood but allowed its
39 kDa counterpart to be excreted in the urine.

Based on their resolved locations in the 2-DE gels and MS/MS derived sequences, the
enhanced albumin spots detected in the urine of ovarian cancer patients in this study
appeared to be fragments of albumin that consist of amino acids between positions
118 to 281, and with an approximate molecular mass of 19 kDa. The human urine is known
to contain low levels of albumin fragments, with some polypeptides containing discontinuous
sequences joined by unknown crosslinks [29]. Since the 19 kDa albumin fragment was present only in trace quantities in the urine
of the control subjects, it may be used as a complementary urine biomarker to differentiate
ovarian carcinoma patients from healthy individuals.

Conclusion

The proteomic profiling of urine samples demonstrated reduced levels of CD59, kininogen-1
and a 39 kDa ITIH4 fragment, as well as the enhanced excretion of a 19 kDa fragment
of albumin in patients with ovarian carcinoma compared to control women. This observation
may be applied in the development of noninvasive protocols for diagnosis and/or monitoring
of the cancer.

Methods

Urine samples and processing

Urine samples were collected from patients newly confirmed with stages II and III
ovarian carcinoma (n = 11), prior to treatment, at the University of Malaya Medical
Centre (UMMC), Kuala Lumpur. All patients showed normal serum creatinine values. Control
urine samples were collected randomly from age-matched cancer negative women (n =
15). Samples obtained were with consent and approval granted by the ethical committee
of UMMC in accordance to the ICH GCP guideline and the Declaration of Helsinki. The
subjects were of different ethnic background (Malay, Chinese and Indian). Sodium azide
was immediately added to the urine upon collection to a final concentration of 20
mM. The samples were centrifuged at 10,000 rpm at 4°C and the supernatant was collected
and dialyzed against distilled water. The urine proteins were aliquoted, freeze-dried
and kept at -20°C. Protein content was determined using the Pierce BCA protein assay
kit (Thermo Fisher Scientific, Rockford USA).

Two-dimensional gel electrophoresis

IPG strips (pH 3-10, 11 cm) were rehydrated overnight in presence of 300 μg urine
proteins in 200 μl rehydration solution (8 M urea, 0.5% v/v Pharmalyte 3-10, 0.5%
v/v NP-40). Isoelectric focusing was performed using the Multiphor™ II Electrophoresis
unit (GE Healthcare, Uppsala, Sweden) for a total of 12001 Vh at 20°C. The samples
were then reduced by incubation of the strips in equilibrium buffer (50 mM Tris-HCl
pH 8.8, 6 M urea, 30% glycerol, 2% SDS) containing 1% w/v DTT for 15 min prior to
SDS-PAGE, and alkylated using 2.5% w/v iodoacetamide in the same equilibrium buffer
for another 15 min. The strips were then laid onto 12.5% polyacrylamide gels and electrophoresis
was performed at 25 mA per gel.

Silver staining and image analysis

The 2-DE gels were developed by silver staining according to the method of Heukeshoven
and Dernick [30] and scanned using the Image Scanner III. For mass spectrometric analysis, staining
of gels was performed in absence of glutaraldehyde. Protein profiles were evaluated
using the ImageMaster™ 2 D Platinum Software (Version 7). Image analysis was restricted
to protein spot clusters that appeared consistently within each cohort of urine samples.
The levels of proteins in each urine sample were evaluated as a percentage of volume
contribution (%vol) to eliminate possible variations due to differential staining.

Mass spectrometry and database search

Protein spots of interest were excised from the silver stained gels and subjected
to in-gel digestion according to the method of Shevchenko et al. [31]. Gel plugs were destained using 50 mM sodium thiosulphate: 15 mM potassium ferricyanide
(1:1; v/v). Proteins in the plugs were reduced with 10 mM DTT in 100 mM ammonium bicarbonate
for 30 min at 60°C, followed by alkylation with 55 mM iodoacetamide in the same solution
for 20 min at RT in dark. The gel plugs were washed with 50% acetonitrile (ACN) in
100 mM ammonium bicarbonate, dehydrated by incubating in 50 μl ACN for 15 min and
left to dry using a speed vac. Proteins were then digested with 7 ng/μl trypsin in
50 mM ammonium bicarbonate overnight at 37°C, extracted twice using 50% ACN and concentrated
using the speed vac. The resulting peptide solutions were desalted and concentrated
using zip-tips (Perfect Pure C18, Eppendorf, Hamburg, Germany). One μl aliquot was
spotted onto a sample plate with 1 μl of matrix solution (α-cyano-4hydroxycinnamic
acid, 10 mg/ml in 70% v/v ACN, 0.1% v/v TFA) and was allowed to air dry.

MALDI mass spectrometry was performed using the Applied Biosystems 4800 Proteomics
Analyser. Spectra were initially acquired in reflecton mode in the mass range of 1000
to 4000 Da. The instrument was then switched to MS/MS (TOF/TOF). Ten strongest peptides
from the MS scan were isolated, fragmented and reaccelerated to measure their masses
and intensities. The data were exported in a format suitable for submission to the
MASCOT database search program (Matrix Science Ltd., London, UK) and searched against
'all entries'. Identification was accepted when ≥ 5 peptide masses matched to a particular
protein (mass error ± 50 ppm - 1 missed cleavage) and the MOWSE score was over the
threshold score at p = 0.05.

SDS-polyacrylamide gel electrophoresis and Western blotting

Urine samples of patients with ovarian carcinoma (n = 11) and control subjects (n
= 15) were separately pooled and subjected to unidimensional SDS-PAGE according to
the method of Laemmli [32]. Gels consisting of 12.5% w/v acylamide were used. Separated proteins were transferred
to nitrocellulose membranes (0.45 μm) using the NovaBlot Kit of Multiphor II Electrophoresis
System (GE Healthcare, Uppsala, Sweden) at 0.8 mA/cm2.

The membranes were blocked with 3% w/v gelatine in Tris-buffered saline (TBS), pH
7.5, for 1 h at RT and washed three times with the same buffer. They were then incubated
for another 1 h in the following HRP-conjugate solutions: (1) anti-human CD59 (Abcam,
Cambridge, UK - Cat. No. ab9182, at 1:5 dilution), (2) anti-human kininogen-1 (Abnova,
Jhongli, Taiwan - Cat. No. H00003827-B01, at 1:500 dilution), (3) anti-albumin (Sigma
Chemical Company, St. Louis, MO USA - Cat. No. A0433, at 1:40 dilution) and (4) champedak
galactose binding (CGB) lectin (0.01 μg/ml) diluted/dissolved in TBST. The use of
the CGB lectin to detect the C-terminal O-glycosylated ITIH4 fragment has been previously
reported [27]. Development of the Western blot was performed using 25 ng 3,3'-diaminobenzidine
and 5 μl 30% v/v H2O2 in 50 ml TBS. The reaction was stopped by washing the membranes with distilled water.

Statistical analysis

All values are presented as mean ± SD. The Student's t-test was used to analyze significance of differences between control subjects and
patients. A p value of less than 0.05 was considered significant.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SSAS carried out the experiments, analyzed the data and drafted the manuscript; BKL
provided the urine samples; OHH contributed to the design of the study and critically
revised the manuscript; ASS planned the study and critically revised the manuscript.
All authors read and approved the final manuscript.

Acknowledgements

This work was funded by research grants from the University of Malaya (Research University
Grant PS153/2008B) and the Ministry of Science, Technology and Innovation, Malaysia
(IRPA grant 12-02-03-2066).

Cordero OJ, De Chiara L, Lemos-González Y, Páez de la Cadena M, Rodríguez-Berrocal FJ: How the measurements of a few serum markers can be combined to enhance their clinical
values in the management of cancer.